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The non-coding Air RNA is required for silencing autosomal imprinted genes
Author: Frank Sleutels, Ronald Zwart, Denise P. Barlow
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".............................................................. The non-coding Air RNA is required for silencing autosomal imprinted genes Frank Sleutels*, Ronald Zwart* & Denise P. Barlow? * Department of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands ? O � AW Institute of Molecular Biology, Billrothstrasse 11, A5020 Salzburg, Austria ............................................................................................................................................................................. In genomic imprinting, one of the two parental alleles of an autosomal gene is silenced epigenetically by a cis-acting mech- anism 1,2 . A bidirectional silencer for a 400-kilobase region that contains three imprinted, maternally expressed protein-coding genes (Igf2r/Slc22a2/Slc22a3) has been shown by targeted deletion to be located in a sequence of 3.7 kilobases 3?5 , which also contains the promoter for the imprinted, paternally expressed non-coding Air RNA 6 . Expression of Air is correlated with repression of all three genes on the paternal allele 5 ; however, Air RNA overlaps just one of these genes in an antisense orientation 6 . Here we show, by inserting a polyadenylation signal that truncates 96% of the RNA transcript, that Air RNA is required for silencing. The truncated Air allele maintains imprinted expression and methylation of the Air promoter, but shows complete loss of silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster on the paternal chromosome. Our results indicate that non-coding RNAs have an active role in genomic imprinting. Most mammalian imprinted genes are found in clusters that also contain imprinted non-coding RNAs 2,7 . In many cases, expression of the non-coding RNA from one parental allele correlates with repression of linked protein-coding genes on the same allele, raising the possibility that non-coding RNAs either are required for silencing or are a consequence of the silencing mechanism. Studies of the Igf2/Ins2/H19 imprinted gene cluster on mouse chromosome 7 have shown, however, that the H19 non-coding RNA does not havearoleinsilencingtheIgf2/Ins2 protein-coding genes 8,9 . Instead, imprinted expression of Igf2/Ins2 occurs through a silencer element 10 and an insulator element (called H19-DMR) close to the H19 promoter, and imprinted expression of H19 is thought to occur as a consequence of paternal-specific methylation of the insulator element 11,12 . Thus, despite the prevalence of imprinted autosomal non-coding RNAs, none has been shown to be required for the silencing of flanking imprinted protein-coding genes. We have studied the paternal-specific Air non-coding RNA on mouse chromosome 17 that is expressed from a promoter located in intron 2 of the Igf2r gene. This non-coding RNA is 108 kilobases (kb), polyadenylated but apparently unspliced, and overlaps the Igf2r promoter 3,6 . Deletion of a 3.7-kb imprint control element (ICE) that contains the Air promoter releases paternal-specific silencing of three genes, Slc22a2 and Slc22a3 that are located upstream, and Igf2r that is located downstream 4,5 . Thus, the 3.7- kb ICE has a bidirectional action despite the fact that Air overlaps only one of these genes (ref. 5; and Fig. 1a). To test whether imprinted expression of the Air RNA is required for silencing, we truncated Air to 4% of its length by inserting a 1.2- kb polyadenylation cassette from the b-globin gene at the down- stream border of the 3.7-kb ICE to yield the Air-T allele (Fig. 1). The modification was designed to truncate Air without disrupting the function of the 3.7-kb ICE, as assessed by its ability to attract a maternal-specific methylation imprint and to show paternal- specific expression of the Air promoter 3,6 . Mice with a maternally inherited Air-T allele (Air-T/ , note that the maternal allele precedes the paternal one in all genotypes shown here) were identical to wild type, whereas mice with a paternally inherited Air-T allele ( /Air-T) and homozygous Air-T mice showed a 15% reduction in birth weight as compared with wild-type littermates (data not shown). A similar phenotype caused by biallelic expression of Igf2r is seen in mice with a paternally inherited deletion of the 3.7-kb ICE; these mice do not express Air owing to 312 Ap Ap Bg Bg BgBB BgM Wild type BgB neo tk S S BgBS SB Targeting vector neo-tk allele Air-T allele BE EMBg SBg Bg Air Air-T S BgBg Bg Sa A C D E Igf2r SB 3.7-kb ICE EME Bg Ap tkneo +/neo-tk+/Air - T +/+ - 9.7 kb - 5.4 kb - 7.0 kb d a c +/++/neo-tk+/neo-tk - 14 kb - 10 kb b 2 kb Slc22a3 Slc22a1 Igf2r Mas PlgSlc22a2 Air 50 kb- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Figure 1 Generation of the Air-T allele. a, Map spanning the 400-kb imprinted cluster. Indicated are paternally expressed Air (hatched box), maternally expressed Igf2r, Slc22a2 and Slc22a3 (dotted boxes) and non-imprinted genes (black boxes). b, Wild- type allele comprises Igf2r (black arrow), Igf2r exons 1?3 (black boxes) and Air (dashed arrow). The locus was targeted at a Bam HI site bordering the 3.7-kb ICE (dotted line) that lies 3.0-kb downstream from the Air transcription start. The targeting vector contains a 1.2-kb rabbit b-globin gene polyadenylation cassette (Ap), and PGK? neomycin (neo) and PGK?thymidine-kinase (tk) selection genes flanked by loxP sites (triangles). Selection genes were deleted from the neo-tk allele by transient Cre transfection to generate the Air-T allele. Probes A, C, D and E are indicated. B, Bam HI; Bg, BglII; E, Eco RI; M, Mlu I; Sa, Sal I; S, ScaI. c, Targeted neo-tk clones were identified by a 10-kb ScaI fragment with probe C. Targeting was paternal-specific in 18 out of 18 ES clones (Supplementary Information). d, The Air-T allele was identified by a 5.4-kb BglII fragment with probe D. letters to nature NATURE | VOL 415 | 14 FEBRUARY 2002 | www.nature.com810 � 2002 Macmillan Magazines Ltd deletion of the Air promoter 4 . We also analysed the Air-T allele in reciprocal, double hetero- zygous crosses with mice containing a 3-cM deletion that spanned this imprinted cluster (T hp ). In a wild-type background, maternal inheritance of the T hp allele (T hp / ) is lethal between 15.5 and 17.5 days postcoitum (d.p.c.), owing to the absence of Igf2r 4,13 .We found, however, that offspring with the T hp /Air-T genotype were normal sized, viable and fertile, indicating that the maternal-specific T hp phenotype had been rescued by the paternal Air-T allele (data not shown), as shown previously in crosses with a paternal 3.7-kb ICE deletion 4,5 . Offspring with the reciprocal genotype (Air-T/T hp ) showed similar size, viability and fertility (data not shown) to a /T hp genotype. We tested that the Air RNA expressed from the Air-T allele was truncated by using an RNase protection assay (RPA) with probes positioned across the 108 kb that is spanned by the endogenous Air RNA 6 (Fig. 2a). Probes downstream from the inserted polyadenyla- tion signal detected Air RNA from the paternal wild-type allele but not from the paternal Air-T allele, indicating the absence of stable Air RNA downstream from the polyadenylation cassette (Fig. 2b). We tested for polyadenylation at the inserted cassette by polymerase chain reaction with reverse transcription (RT-PCR) using a poly(dT) reverse transcription primer plus the PX6R gene-specific primer (Fig. 2a). Unexpectedly, RT-PCR generated a single 314- base-pair (bp) Air-T-specific fragment from a spliced, polyadeny- lated RNA that had used a splice donor located 53-bp downstream from the principal Air transcription start and a splice acceptor from the inserted exon 3 of the b-globin gene (Supplementary Infor- mation). The 53-bp splice donor has been identified in a transgene study but is not normally used in the wild-type locus; however, use of this splice donor does not correlate with the loss of imprinting of Igf2r/Air in transgenes 6,14 . We investigated the proportion of spliced/unspliced Air-T RNA by RPA with probes positioned between the Air promoter and the inserted polyadenylation cassette (Fig. 2c). Both probe G and probe J showed that 20% unspliced Air RNA was present from the paternal Air-T allele as compared with the paternal wild-type allele (Fig. 2c). Probe K (comprising the RT-PCR fragment) detected both spliced and unspliced RNAs, and confirmed that these were present at 80% and 20%, respectively. Probe L spans the inserted polyadenylation signal and extends 404-bp downstream from the ATTAAA signal; this probe detected the same 173-bp protected fragment as found in the control LAI-313 transgene 14 , showing that the ATTAAA signal is used, but not larger protected fragments (Fig. 2c). Thus, Fig. 2 shows that although the Air-T allele generates spliced and unspliced Air RNAs, these are both truncated 4.2-kb downstream from the Air promoter and stable RNA is not detected downstream from the polyadenylation cassette. We tested the function of the 3.7-kb ICE in the Air-T allele by analysing the methylation state, imprinted expression and the expression level of the Air promoter in Air-T mice as compared with wild-type littermates. Methyl-sensitive enzymes identified methylation specific to the maternal allele on the Air-T promoter as found on the wild-type allele (Fig. 3a). Analysis by RPA showed imprinted, paternal-specific expression of the Air-T RNA as found on the wild-type allele, and also showed that the Air-T allele generates similar levels of stable Air RNA as compared with the wild-type allele (Fig. 3b). Although it is possible that insertion of the polyadenylation cassette disturbed an as yet unidentified imprinting element or altered transcription/stability of the Air-T RNA, the - Air (47 bp) T hp / Air -T T hp /+ +/ T hp PC Probe K - Air (240 bp) LAI-313 - Air (287 bp) - Aprt (134 bp) - Aprt (134 bp) T hp / Air -T T hp /+ +/ T hp PC Probe L - poly(A) (173 bp) LAI-313 T hp /Air -T T hp /+ +/ T hp PC Probe G - (207 bp) - (171 bp) Air - (148 bp) - (134 bp) Aprt Probe J T hp / Air -T T hp /+ +/ T hp PC - Aprt (134 bp) - Air (155 bp) c * a Ap 1 Igf2r 2 Mas RT F G M Air PX6R K L JC 3 B DHI Probe I T hp / Air -T +/+ +/ T hp PC - Aprt (134 bp) - Air (196 bp) - Aprt (134 bp) - Air (185 bp) T hp / Air -T +/+ T hp /+ +/ T hp PC Probe BProbe H - Aprt (134 bp) - Air (221 bp) T hp / Air -T +/+ +/ T hp PC b Figure 2 The Air-T allele truncates the Air RNA. a, Position of RNA probes (B?D, F?J, K (intron spanning), L), and RT and PX6R oligonucleotides used in RT-PCR, relative to those of Mas, Igf2r (exons 1?3) and Air on the Air-T allele. Unspliced Air-T, thin arrow; spliced Air-T, dashed arrow; Air-T exon 1, black box upstream from the Mlu I (M) site; polyadenylation cassette, Ap. b, RPA with probes B (on 11.5-d.p.c. embryo RNA), H and I (on adult heart RNA) indicates the absence of Air at, respectively, 28, 41 and 95 kb downstream from Ap. Identical results were obtained with probes C and D (Supplementary Information). c, RPA with probes G and J?L on 11.5-d.p.c. embryo RNA shows expression of truncated spliced and unspliced Air-T RNA. Probe G (which overlaps a 53-bp splice donor and multiple Air transcription start sites) protects 207, 171 and 148 bp of unspliced Air RNA from the Air-T paternal allele. However, the signal quantified by phosphorimager was reduced by 80% compared with that of the wild-type paternal allele (note the relative loading of Air-T and T hp / and that spliced fragments protected by probe G are not visible). The signal reduction for unspliced Air-T is confirmed by probe J (which is located in the intron of the spliced Air-T RNA). Probe K is an RT-PCR fragment generated with oligonucleotides RT and PX6R (Supplementary Information) and protects 287 bp of spliced and 240 bp plus 47 bp of unspliced Air-T RNA (the 240-bp fragment is specific for the Ap cassette; asterisk indicates a faint 47- bp Air fragment). Probe K shows a ratio of 80% spliced and 20% unspliced Air-T RNA. The multicopy transgene LAI-313 contains the identical polyadenylation cassette 14 , and more clearly protects the 240- and 47-bp unspliced Air fragments. Probe L spans the AATAAA polyadenylation signal and protects 173 bp of T hp /Air-T and LAI-313. The absence of larger, protected probe-L fragments confirms that both the spliced and unspliced Air-T RNAs are truncated at the polyadenylation signal. In b and c, parental alleles are denoted maternal/paternal, the loading control is Aprt exon 3, input probes are indicated by P, and the tRNA control by C. letters to nature NATURE | VOL 415 | 14 FEBRUARY 2002 | www.nature.com 811� 2002 Macmillan Magazines Ltd observation that the Air-T promoter was expressed and imprinted in a similar manner to that of the Air wild-type promoter indicates that function of the 3.7-kb ICE was not disturbed. We examined the effect of the truncated Air-T RNA on paternal silencing of the imprinted cluster. Of the three genes in this cluster, only Igf2r becomes methylated when silenced on the wild-type paternal allele 5 . Methylation of Igf2r is completely absent from the paternal Air-T allele in contrast to the wild-type allele (Fig. 4a). We used RNA blots to assess imprinted expression and show (Fig. 4b, c) that all three genes, Igf2r, Slc22a2 and Slc22a3, have a complete loss of silencing on the paternal Air-T allele. Maternal expression of all three genes from the Air-T allele was at the same level as maternal expression from the wild-type allele, indicating that the targeting event did not alter their expression. The Air-T allele truncated the Air RNA to 4% of its length yet maintained the imprinted status and expression level of the wild- type paternal Air promoter (with the caveats noted above). This truncation caused a complete loss of silencing of the Igf2r/Slc22a2/ Slc22a3 cluster on the paternal chromosome. This shows that the Air non-coding RNA is required for cis repression of flanking protein- coding genes. The identification of a role for the Air non-coding RNA in cis repression contrasts with the previous observation that DNA insulator and silencer elements regulate imprinting of the Ins2/Igf2/H19 cluster 10 ? 12 . Our results thus identify a new RNA- dependent mechanism in genomic imprinting. The bidirectional action of the Air RNA shown by our data is unexpected because the wild-type Air RNA overlaps only one gene. Repression, at least of Slc22a2 and Slc22a3, does not require transcriptional overlap, yet overlap with non-coding RNAs has been found at five independent imprinted loci 3,15 ? 18 . It is possible that the transcriptional overlap by non-coding RNAs may be involved if silencing of the non-overlapped genes were secondary to silencing of the overlapped gene. In this two-step model, the antisense non-coding RNA might repress the overlapped promoter (for example, by promoter occlusion or a form of cis-acting RNA interference; note that expression?competition 3 is excluded by our findings) and induce a silent chromatin state that could spread bidirectionally in a limited manner and silence flanking genes. Alternatively, if transcriptional overlap is not involved in cis repression, then the non-coding RNAs might function to recruit repressor proteins to the gene cluster. This latter model has parallels with X inactivation in female mammals, in which expression of the non-coding Xist RNA inactivates one X chromosomes in cis 19 . The Xist RNA has features that have not been characterized as yet or that might be absent from imprinted non-coding RNAs, such as the ability to coat and spread along the length of the repressed chromosome 20 ? 23 . Although further testing is clearly necessary to determine whether there are mechanistic similarities at the mo- lecular level, it is notable that X inactivation has been suggested to have evolved from a localized form of imprinting that initially b PC - Aprt (134 bp) - Air (47 bp) T hp / Air - T Air - T / T hp +/+ T hp /+ +/ T hp Probe F T hp / Air - T - + - + - + - + - + Air - T / T hp +/+ T hp /+ +/ T hp - 6.3 kb - 5.1 kb - 3.6 kb - 4.8 kb a Figure 3 The Air-T allele is imprinted. a, The Air-T allele retains a methylation imprint on the Air promoter. DNA from 11.5-d.p.c. embryos cut with Eco RI (-) or Eco RI/Mlu I( ) was analysed with probe E. The fragments correspond to wild type methylated, 6.3 kb; wild type unmethylated, 5.1 kb; Air-T methylated, 4.8 kb; and Air-T unmethylated, 3.6 kb. Identical results were obtained with adult tail and spleen, and Sfu I (Supplementary Information). b, The Air-T allele maintains imprinted Air expression. RPA was carried out on 11.5-d.p.c. embryo RNA with probe F, which detects spliced and unspliced Air. The paternal Air-T and wild-type alleles show equal Air expression, whereas the maternal alleles repress Air. Identical results were obtained with heart RNA and probes G, J and K (Supplementary Information). P, input probes; C, tRNA control. b T hp / Air - T ? + ? + ? + ? + Air - T / T hp +/+ +/ T hp - 5.5 kb - 9.5 kb a T hp / Air - T Air - T / T hp +/+ +/ T hp T hp /+ - Gapd 1.3 kb - Slc22a3 3.5 kb - Slc22a2 2.2 kb c - Igf2r 9 kb T hp / Air - T Air - T / T hp +/+ +/ T hp T hp /+ - Gapd 1.3 kb Figure 4 Loss of paternal silencing on the Air-T allele. a, The Igf2r promoter on the Air-T allele lacks paternal methylation. Tail DNA was cut with Bgl II (-) or BglI/Sal I( ) and analysed with probe A. Methylated fragment, 9.5 kb; unmethylated fragment, 5.5 kb. Identical results were obtained with adult spleen DNA and either Not IorSmaI (Supplementary Information). b, Loss of paternal Igf2r repression on the Air-T allele. RNA from 11.5-d.p.c. embryos was analysed with a probe covering exons 3?6 from Igf2r. Identical results were obtained with adult heart RNA (Supplementary Information). c, Loss of paternal Slc22a2 and Slc22a3 repression on the Air-T allele. RNA from 11.5-d.p.c. placenta was analysed with cDNA probes for Slc22a2 and Slc22a3.Inb and c, Gapd is a control for RNA loading. letters to nature NATURE | VOL 415 | 14 FEBRUARY 2002 | www.nature.com812 � 2002 Macmillan Magazines Ltd affected only a small region of the X chromosome that contains a dosage-sensitive gene 24,25 . A Methods Generation of the Air-T allele The targeting vector was constructed from a 9.6-kb BglII fragment (bp 117,878?127,575; AJ249895). A selection cassette containing neomycin and the thymidine kinase gene, each driven by a PGK promoter flanked by loxP sites plus a 1.2-kb fragment from the rabbit b- globin gene (bp 31,392?32,590; M18818) containing part of exon 2, complete intron 3 and complete exon 3 with the polyadenylation signal in the correct orientation for the Air promoter, was inserted at the BamHI site in this fragment. The selection cassette was deleted by electroporation of a plasmid encoding Cre recombinase 26 and transient puromycin selection. We generated chimaeric mice by injecting paternally targeted Air-T embryonic stem (ES) cells into C57/Bl6 blastocysts 27 . Mice carrying the Air-T allele were maintained on an FVB/N background. Methylation and expression analyses Digestion of methyl-sensitive enzymes was monitored by hybridization to mitochondrial DNA. We carried out RPA using an RPAIII kit (Ambion). Signals were quantified with a Phosphorimager (Fujix). For RT-PCR, 5 mg heart RNA was reverse transcribed with Superscript (Gibco) using the linker-poly(dT) primer RT, 5 0 -CTGGGAAACAGCTATGA CCATGATCGATTTTTTTTTTTTTTTTTN-3 0 , and amplified with PX6R, 5 0 -GAAGCAC AGCACCGCCAGTTAC-3 0 , for 30 cycles (94 8C, 30 s; 59 8C, 30 s; 72 8C, 120 s). Probes for DNA analyses We used the following probes: probe A, a 325-bp PCR fragment (bp 102,813?103,137; AJ249895); probe C, a 0.9-kb EcoRI/HinCII fragment from EST AA592338 (ref. 6) collinear with genomic DNA; probe D, (bp 121,834?122,862; AJ249895); and probe E (bp 124,992?126,086; AJ249895). Probes for RNA analyses Probe B, described as probe GFPAIR 14 , is 322 bp and protects 185 bp of Air RNA at the Igf2r promoter. Probes C and D are the same as the DNA analyses probes. Probe F is 174 bp and protects 47 bp (bp 126,181?126,227; AJ249895) immediately downstream from the principal Air transcription start site 6 . Probe G (bp 126,086?126,293; AJ249895), described as probe MlMs1 (refs 6,14), detects unspliced (207, 171 and 148 bp) Air RNA fragments. This relatively large probe did not produce a clear signal for the spliced Air-T RNA (predicted fragments of 112, 76 and 53 bp), which should have been recognized (see probe K below). Probe H, described as probe RPA1 (ref. 6), protects 221 bp of Air RNA (bp 85,250?85,029; AJ249895). Probe I, described as probe MS1B8 (ref. 6) protects 196 bp. Probe J is 173 bp and protects 155 bp of Air RNA (bp 123,233-123,387; AJ249895). Probe K is 364 bp, contains the RT-PCR fragment made with probes RT and PX6R (Supplementary Information) and protects 287 bp of spliced and 240 and 47 bp of unspliced polyadenylated RNA. Probe L spans 558 bp (bp 32,032?32,590; M18818) of the b-globin gene (including the polyadenylation signal) and protects 173 bp of polyadenylated RNA (bp 32,032?32,204; M18818). Probe L extends 385 bp downstream from the polyadenylation signal and the absence of protected fragments longer than 173 bp indicates use of the polyadenylation signal. The Aprt template is a 252-bp XhoI/ XbaI fragment (bp 2,165?2,417; M11310) and protects Aprt exon 3 (134 bp). For RNA blots, we used the following probes: for Igf2r, complementary DNA exons 3?6; for Slc22a2, bp 989?1,605 (AJ006036); for Slc22a3 bp 1?2,766 (AF078750); for Gapd, complete cDNA. Received 27 September; accepted 4 December 2001. 1. Reik, W. & Walter, J. Genomic imprinting: parental influence on the genome. Nature Rev. Genet. 2, 21?32 (2001). 2. Sleutels, F. & Barlow, D. P. in Homology Effects (eds Wu, C.-t. & Dunlap, C.) (Academic, San Diego, in the press). 3. Wutz, A. et al. Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature 389, 745?749 (1997). 4. Wutz, A. et al. Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice. Development 128, 1881?1887 (2001). 5. Zwart, R., Sleutels, F., Wutz, A., Schinkel, A. H. & Barlow, D. P. Bidirectional action of the Igf2r imprint control element on upstream and downstream imprinted genes. Genes Dev. 15, 2361?2366 (2001). 6. Lyle, R. et al. The imprinted antisense RNA at the Igf2r locus overlaps but does not imprint Mas1. Nature Genet. 25, 19?21 (2000). 7. Beechey, C. V., Cattanach, B. M. & Selley, R. L. Mouse Imprinting Data and References. MRC Mammalian Genetics Unit [online] khttp://www.mgu.har.mrc.ac.uk/imprinting/imprinting.htmll (2000). 8. Schmidt, J. V., Levorse, J. M. & Tilghman, S. M. Enhancer competition between H19 and Igf2 does not mediate their imprinting. Proc. Natl Acad. Sci. USA 96, 9733?9738 (1999). 9. Hark, A. T. et al. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405, 486?489 (2000). 10. Constancia, M. et al. Deletion of a silencer element in Igf2 results in loss of imprinting independent of H19. Nature Genet. 26, 203?206 (2000). 11. Bell, A. C. & Felsenfeld, G. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405, 482?485 (2000). 12. Reik, W. & Murrell, A. Genomic imprinting. Silence across the border. Nature 405, 408?409 (2000). 13. Wang, Z. Q., Fun, M. R., Barlow, D. P. & Wagner, E. F. Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene. Nature 372, 464?467 (1994). 14. Sleutels, F. & Barlow, D. P. Investigation of elements sufficient to imprint the mouse Air promoter. Mol. Cell. Biol. 21, 5008?5017. (2001). 15. Wroe, S. F. et al. An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus. Proc. Natl Acad. Sci. USA 97, 3342?3346 (2000). 16. Lee, Y. J. et al. Mit1/Lb9 and Copg2, new members of mouse imprinted genes closely linked to Peg1/ Mest 1 . FEBS Lett. 472, 230?234 (2000). 17. Rougeulle, C., Cardoso, C., Fontes, M., Colleaux, L. & Lalande, M. An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nature Genet. 19, 15?16 (1998). 18. Smilinich, N. J. et al. A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome. Proc. Natl Acad. Sci. USA 96, 8064?8069 (1999). 19. Avner, P. & Heard, E. X-chromosome inactivation: counting, choice and initiation. Nature Rev. Genet. 2, 59?67 (2001). 20. Clemson, C. M., McNeil, J. A., Willard, H. F. & Lawrence, J. B. XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132, 259?275 (1996). 21. Lee, J. T., Strauss, W. M., Dausman, J. A. & Jaenisch, R. A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell 86, 83?94 (1996). 22. Sheardown, S. A. et al. Stabilization of Xist RNA mediates initiation of X chromosome inactivation. Cell 91, 99?107 (1997). 23. Wutz, A. & Jaenisch, R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol. Cell 5, 695?705 (2000). 24. Lyon, M. F. Imprinting and X-chromosome inactivation. Results Probl. Cell Differ. 25, 73?90 (1999). 25. Graves, J. A. Mammals that break the rules: genetics of marsupials and monotremes. Annu. Rev. Genet. 30, 233?260 (1996). 26. O?Gorman, S., Dagenais, N. A., Qian, M. & Marchuk, Y. Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells. Proc. Natl Acad. Sci. USA 94, 14602?14607 (1997). 27. Hogan, B. L. M., Beddington, R. S. P., Costantini, F. & Lacy, E. Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1994). Supplementary Information accompanies the paper on Nature?s website (http://www.nature.com). Acknowledgements We thank K. van Veen, K. van het Wout, P. Krimpenfort for help in generating mice; S. Greven, T. Maidment and N. Bosnie for care of the mice; A. Berns, H. te Riele, M. van Lohuizen, R. Beijersbergen, P. Borst and A. Frischauf for comments; and A. Berns for help and encouragement. This research was supported by the Dutch Cancer Society (KWF). Correspondence and requests for materials should be addressed to D.P.B. (e-mail: dbarlow@imb.oeaw.ac.at). .............................................................. Structural basis for antagonist- mediated recruitment of nuclear co-repressors by PPARa H. Eric Xu, Thomas B. Stanley, Valerie G. Montana, Millard H. Lambert, Barry G. Shearer, Jeffery E. Cobb, David D. McKee, Cristin M. Galardi, Kelli D. Plunket, Robert T. Nolte, Derek J. Parks, John T. Moore, Steven A. Kliewer, Timothy M. Willson & Julie B. Stimmel Nuclear Receptor Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA ............................................................................................................................................................................. Repression of gene transcription by nuclear receptors is mediated by interactions with co-repressor proteins such as SMRT and N- CoR 1,2 , which in turn recruit histone deacetylases to the chro- matin 3?5 . Aberrant interactions between nuclear receptors and co-repressors contribute towards acute promyelocytic leukaemia and thyroid hormone resistance syndrome 6?8 . The binding of co- repressors to nuclear receptors occurs in the unliganded state, and can be stabilized by antagonists 9 . Here we report the crystal structure of a ternary complex containing the peroxisome pro- liferator-activated receptor-a ligand-binding domain bound to the antagonist GW6471 and a SMRT co-repressor motif. In this structure, the co-repressor motif adopts a three-turn a-helix that prevents the carboxy-terminal activation helix (AF-2) of the receptor from assuming the active conformation. Binding of letters to nature NATURE | VOL 415 | 14 FEBRUARY 2002 | www.nature.com 813� 2002 Macmillan Magazines Ltd "
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Cell Cycle and Cell Division
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Career Planning
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